Prion protein (PrPC) is a 33-35 kD protein of uncertain function and, in humans, is transcribed by a gene on the short arm of chromosome 20. The 27-30 kD protease-resistant core (prion, scrapie protein, or PrPSc) is the functional component, with several isoforms responsible for “prion diseases,” which are protein conformational diseases.
Protein conformational diseases arise from aberrant conformational transition of a protein (a conformational disease protein such as PrPC), which in turn leads to self-association of the aberrant protein forms (e.g., PrPSc) resulting in tissue deposition and damage. Prions (PrPSc) have a substantially pleated sheet conformation rather than the α-helix structure of normal PrPC, lack detectable nucleic acid, and generally do not elicit an immune response. In general, protein conformational diseases share striking similarities in clinical presentations, typically a rapid progression from diagnosis to death following varying lengths of incubation.
In humans, prion diseases, also known as, “transmissible spongiform encephalopathies” (TSEs), include, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), Fatal Familial Insomnia, and Kuru (see, e.g., Isselbacher et al., eds. (1994). Harrison's Principles of Internal Medicine. New York: McGraw-Hill, Inc.; Medori et al. (1992) N. Engl. J. Med. 326: 444-9). In animals, TSEs include sheep scrapie, bovine spongiform encephalopathy (BSE), transmissible mink encephalopathy, and chronic wasting disease of captive mule deer and elk (Gajdusek, (1990). Subacute Spongiform Encephalopathies: Transmissible Cerebral Amyloidoses Caused by Unconventional Viruses. In: Virology, Fields, ed., New York: Raven Press, Ltd. (pp. 2289-2324)). Transmissible spongiform encephalopathies are characterized by the same hallmarks: the presence of the abnormal (beta-rich, proteinase K resistant) conformation of the prion protein that transmits disease when experimentally inoculated into laboratory animals including primates, rodents, and transgenic mice.
Recently, the rapid spread of BSE and its correlation with elevated occurrence of TSEs in humans has led to increased interest in the detection of TSEs in non-human mammals. The tragic consequences of accidental transmission of these diseases (see, e.g., Gajdusek, Infectious Amyloids, and Prusiner Prions In Fields Virology. Fields, et al., eds. Philadelphia: Lippincott-Ravin, Pub. (1996); Brown et al. Lancet, 340: 24-27 (1992)), decontamination difficulties (Asher et al. (1986) In: Laboratory Safety: Principles and Practices, Miller ed., (pp. 59-71) Am. Soc. Micro.), and concern about BSE (British Med. J. (1995) 311: 1415-1421) underlie the urgency of having both a diagnostic test that would identify humans and animals with TSEs and therapies for infected subjects.
Prions differ significantly from bacteria, viruses and viroids. The dominating hypothesis is that, unlike all other infectious pathogens, infection is caused by an abnormal conformation of the prion protein, which acts as a template and converts normal prion conformations into abnormal, aberrant conformations. A prion protein was first characterized in the early 1980s. (See, e.g., Bolton, McKinley et al. (1982) Science. 218: 1309-1311; Prusiner, Bolton et al. (1982) Biochemistry 21: 6942-6950; McKinley, Bolton et al. (1983) Cell 35: 57-62). Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. (See, e.g., Basler, Oesch et al. (1986) Cell 46: 417-428.)
The key characteristic of prion diseases is the formation of the abnormally shaped protein (PrPSc) from the normal form of prion protein (cellular or nonpathogenic or PrPC). (See, e.g., Zhang et al. (1997) Biochem. 36(12): 3543-3553; Cohen & Prusiner (1998) Ann. Rev. Biochem. 67: 793-819; Pan et al. (1993) Proc. Natl. Acad. Sci. USA 90:10962-10966; Safar et al. (1993) J Biol. Chem. 268: 20276-20284.) The substantially β-sheet structure of PrPSc as compared to the predominantly α-helical folded non-disease forms of PrPC has been revealed by optical spectroscopy and crystallography studies. (See, e.g., Wille et al. (2001) Proc. Nat'l Acad. Sci. USA 99: 3563-3568; Peretz et al. (1997) J. Mol. Biol. 273: 614-622; Cohen & Prusiner, (1999) 5: Structural Studies of Prion Proteins. In Prion Biology And Diseases, S. Prusiner, ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. (pp: 191-228.) The structural changes appear to be followed by alterations in biochemical properties: PrPC is soluble in non- denaturing detergents, PrPSc is insoluble; PrPC is readily digested by proteases, while PrPSc is partially resistant, resulting in the formation of an amino-terminally truncated fragment known as “PrPres” (Baldwin et al. (1995); Cohen & Prusiner (1995)), “PrP 27-30” (27-30 kDa) or “PK-resistant” (proteinase K resistant) form. Additionally, PrPSc can convert PrPC to the pathogenic conformation. See, e.g., Kaneko et al. (1995) Proc. Nat'l Acad. Sci. USA 92:11160-11164; Caughey (2003) Br Med Bull. 66: 109-20.
Detection of the pathogenic isoforms of conformational disease proteins in living subjects, and samples obtained from living subjects, has proven difficult. Thus, definitive diagnosis and palliative treatments for these transmissible and amyloid-containing conditions before death of the subject remains a substantially unmet challenge. Histopathological examination of brain biopsies is risky to the subject and lesions and amyloid deposits can be missed depending on where the biopsy sample is taken from. Also, there are still risks involved with biopsies to animals, patients, and health care personnel. Further, the results from brain tests on animals are not usually obtained until the animal has entered the food supply. Also, typically, antibodies generated against prion peptides recognize both denatured PrPSc and PrPC but are unable to selectively recognize infectious (undenatured) PrPSc. (See, e.g., Matsunaga et al. (2001) Proteins: Structure, Function and Genetics 44: 110-118).
A number of tests for TSE are available (See, Soto, C. (2004) Nature Reviews Microbiol. 2:809, Biffiger et al. (2002) J. Virol. Meth. 101:79; Safar et al. (2002) Nature Biotech. 20:1147, Schaller et al. Acta Neuropathol. (1999) 98:437, Lane et al. (2003) Clin. Chem. 49:1774). However, all of these utilize brain tissue samples and are suitable only as post-mortem tests. Most of these require proteinase K treatment of the samples as well, which can be time-consuming, incomplete digestion of PrPC can lead to false positive results, and digestion of PK-sensitive PrPSC can yield false negative results.
Thus, there remains a need for compositions and methods for detecting the presence of the pathogenic prion proteins in various samples, for example in samples obtained from living subjects, in blood supplies, in farm animals and in other human and animal food supplies. There also remains a need for methods and compositions for diagnosing and treating prion-related diseases. This invention is directed to these, as well as other, important ends.